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1.
Chem Commun (Camb) ; 55(63): 9311-9314, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31310244

RESUMO

Discrimination between cysteine and homocysteine at the single-molecule level is achieved within a K238Q mutant aerolysin nanopore, which provides a confined space for high spatial resolution to identify the amino acid difference with a 5'-benzaldehyde poly(dA)4 probe. Our strategy allows potential detection and characterization of various amino acids and their modifications, and provides a crucial step towards developing nanopore protein sequencing devices.


Assuntos
Toxinas Bacterianas/química , Cisteína/análise , Homocisteína/análise , Nanoporos , Proteínas Citotóxicas Formadoras de Poros/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Cromatografia Líquida de Alta Pressão , Mutagênese Sítio-Dirigida , Poli A/química , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Espectrometria de Massas por Ionização por Electrospray
2.
Nat Commun ; 10(1): 2900, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31263098

RESUMO

The alpha helical CytolysinA family of pore forming toxins (α-PFT) contains single, two, and three component members. Structures of the single component Eschericia coli ClyA and the two component Yersinia enterolytica YaxAB show both undergo conformational changes from soluble to pore forms, and oligomerization to produce the active pore. Here we identify tripartite α-PFTs in pathogenic Gram negative bacteria, including Aeromonas hydrophila (AhlABC). We show that the AhlABC toxin requires all three components for maximal cell lysis. We present structures of pore components which describe a bi-fold hinge mechanism for soluble to pore transition in AhlB and a contrasting tetrameric assembly employed by soluble AhlC to hide their hydrophobic membrane associated residues. We propose a model of pore assembly where the AhlC tetramer dissociates, binds a single membrane leaflet, recruits AhlB promoting soluble to pore transition, prior to AhlA binding to form the active hydrophilic lined pore.


Assuntos
Aeromonas hydrophila/metabolismo , Toxinas Bacterianas/química , Proteínas Hemolisinas/química , Proteínas Citotóxicas Formadoras de Poros/química , Aeromonas hydrophila/química , Aeromonas hydrophila/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Cristalografia por Raios X , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo
3.
J Microbiol Biotechnol ; 29(7): 1014-1021, 2019 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-31216790

RESUMO

In the bacterial community, unicellular organisms act together as a multicellular being. Bacteria interact within the community and programmed cell death (PCD) in prokaryotes is a sort of altruistic action that enables the whole population to thrive. Genetically, encoded cell death pathways are triggered by DNA damage or nutrient starvation. Given the environmental and bacterial diversity, different PCD mechanisms are operated. Still, their biochemical and physiological aspects remain unrevealed. There are three main pathways; thymineless death, apoptosis-like death, and toxin-antitoxin systems. The discovery of PCD in bacteria has revealed the possibility of developing new antibiotics. In this review, the molecular and physiological characteristics of the three types of PCD and their development potential as antibacterial agents are addressed.


Assuntos
Apoptose , Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/metabolismo , Viabilidade Microbiana , Timina/metabolismo , Antitoxinas/metabolismo , Bactérias/citologia , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/genética , Toxinas Bacterianas/metabolismo , Dano ao DNA , Resposta SOS (Genética)
4.
J Med Microbiol ; 68(7): 1118-1128, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31172910

RESUMO

PURPOSE: This study investigated the efficacy of the essential mineral, selenium (sodium selenite), in reducing the toxin production, spore outgrowth and antibiotic resistance of Clostridium difficile in vitro. METHODOLOGY: Two hypervirulent C. difficile isolates were cultured in brain heart infusion broth with and without a sub-minimum inhibitory concentration (sub-MIC) of sodium selenite, and the supernatant and bacterial pellet were harvested for total toxin quantitation and RT-qPCR analysis of toxin-encoding genes, respectively. Additionally, C. difficile isolates were cultured in brain heart infusion broth containing 0.5 or 1× the minimum inhibitory concentration (MIC) of either ciprofloxacin or vancomycin with or without sub-MICs of sodium selenite. Further, the effect of sodium selenite on C. difficile germination and spore outgrowth was also determined by exposing C. difficile spores to a sub-MIC of sodium selenite in a germination medium and measuring the germination and outgrowth by measuring the optical density at 600 nm. RESULTS: Sodium selenite significantly reduced C. difficile toxin synthesis, cytotoxicity and spore outgrowth. Further, the expression of the toxin production genes, tcdA and tcdB, was downregulated in the presence of sodium selenite, while sodium selenite significantly increased the sensitivity of C. difficile to ciprofloxacin , but not vancomycin, as revealed by decreased bacterial growth in samples containing ciprofloxacin+selenium compared to the antibiotic control. Although the sub-MIC of sodium selenite did not inhibit spore germination, it was capable of completely inhibiting spore outgrowth. CONCLUSION: Our results suggest that sodium selenite could potentially be used to control C. difficile and indicate that future in vivo studies are warranted.


Assuntos
Antibacterianos/farmacologia , Toxinas Bacterianas/metabolismo , Clostridium difficile/efeitos dos fármacos , Selenito de Sódio/farmacologia , Esporos Bacterianos/efeitos dos fármacos , Oligoelementos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clostridium difficile/patogenicidade , Clostridium difficile/fisiologia , Farmacorresistência Bacteriana , Enterotoxinas/genética , Enterotoxinas/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Esporos Bacterianos/fisiologia , Virulência
5.
Nat Commun ; 10(1): 2641, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201325

RESUMO

Epsilon toxin (Etx), a potent pore forming toxin (PFT) produced by Clostridium perfringens, is responsible for the pathogenesis of enterotoxaemia of ruminants and has been suggested to play a role in multiple sclerosis in humans. Etx is a member of the aerolysin family of ß-PFTs (aß-PFTs). While the Etx soluble monomer structure was solved in 2004, Etx pore structure has remained elusive due to the difficulty of isolating the pore complex. Here we show the cryo-electron microscopy structure of Etx pore assembled on the membrane of susceptible cells. The pore structure explains important mutant phenotypes and suggests that the double ß-barrel, a common feature of the aß-PFTs, may be an important structural element in driving efficient pore formation. These insights provide the framework for the development of novel therapeutics to prevent human and animal infections, and are relevant for nano-biotechnology applications.


Assuntos
Toxinas Bacterianas/química , Clostridium perfringens/ultraestrutura , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Toxinas Bacterianas/metabolismo , Biotecnologia/métodos , Linhagem Celular , Infecções por Clostridium/microbiologia , Infecções por Clostridium/prevenção & controle , Clostridium perfringens/genética , Clostridium perfringens/metabolismo , Clostridium perfringens/patogenicidade , Microscopia Crioeletrônica , Cães , Enterotoxemia/microbiologia , Enterotoxemia/prevenção & controle , Modelos Moleculares , Mutagênese Sítio-Dirigida , Nanotecnologia/métodos , Conformação Proteica em Folha beta/genética , Multimerização Proteica/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
6.
Nat Commun ; 10(1): 2712, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221971

RESUMO

Clostridium difficile (C. difficile) incidence has tripled over the past 15 years and is attributed to the emergence of hypervirulent strains. While it is clear that C. difficile toxins cause damaging colonic inflammation, the immune mechanisms protecting from tissue damage require further investigation. Through a transcriptome analysis, we identify IL-33 as an immune target upregulated in response to hypervirulent C. difficile. We demonstrate that IL-33 prevents C. difficile-associated mortality and epithelial disruption independently of bacterial burden or toxin expression. IL-33 drives colonic group 2 innate lymphoid cell (ILC2) activation during infection and IL-33 activated ILC2s are sufficient to prevent disease. Furthermore, intestinal IL-33 expression is regulated by the microbiota as fecal microbiota transplantation (FMT) rescues antibiotic-associated depletion of IL-33. Lastly, dysregulated IL-33 signaling via the decoy receptor, sST2, predicts C. difficile-associated mortality in human patients. Thus, IL-33 signaling to ILC2s is an important mechanism of defense from C. difficile colitis.


Assuntos
Clostridium difficile/imunologia , Enterocolite Pseudomembranosa/imunologia , Imunidade Inata , Interleucina-33/metabolismo , Linfócitos/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antibacterianos/efeitos adversos , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/metabolismo , Clostridium difficile/patogenicidade , Colo/citologia , Colo/imunologia , Colo/microbiologia , Colo/patologia , Modelos Animais de Doenças , Enterocolite Pseudomembranosa/microbiologia , Enterocolite Pseudomembranosa/mortalidade , Enterocolite Pseudomembranosa/terapia , Transplante de Microbiota Fecal , Feminino , Microbioma Gastrointestinal/efeitos dos fármacos , Microbioma Gastrointestinal/imunologia , Perfilação da Expressão Gênica , Humanos , Interleucina-33/imunologia , Linfócitos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologia , Virulência/imunologia , Adulto Jovem
7.
Pestic Biochem Physiol ; 157: 219-229, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31153472

RESUMO

Photorhabdus akhurstii can produce a variety of proteins that aid this bacterium and its mutualistic nematode vector, Heterorhabditis indica to kill the insect host. Herein, we characterized (by heterologously expressing in E. coli) an open reading frame (1713 bp) of the toxin complex protein, TcaB from P. akhurstii strains IARI-SGHR2 and IARI-SGMS1 and assessed its toxic effect on G. mellonella larvae. The intra-hemocoel injection of purified TcaB (molecular weight-63 kDa) caused fourth instar larval bodies to blacken and die with LD50 values of 67.25 (IARI-SGHR2) and 52.08 (IARI-SGMS1) ng per larva at 12 h. Additionally, oral administration of the toxin caused larval mortality with LD50 values of 709.55 (IARI-SGHR2) and 598.44 (IARI-SGMS1) ng per g diet per larva at 7 days post feeding. Injection of purified TcaB caused loss of viability of fourth instar G. mellonella hemocytes at 6 h post incubation; cells displayed morphological changes typical of apoptosis, including cell shrinkage, membrane blebbing, nuclear condensation and disintegration. Injection of TcaB also elevated the phenoloxidase activity in insect hemolymph which triggers an extensive immune response that potentially leads to larval death. Similar to other bacterial toxins TcaB possesses potent biological activity which may enable it to be used as an efficient agent for pest management.


Assuntos
Proteínas de Bactérias/farmacologia , Toxinas Bacterianas/farmacologia , Inseticidas/metabolismo , Inseticidas/farmacologia , Mariposas/efeitos dos fármacos , Photorhabdus/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Hemócitos/efeitos dos fármacos , Photorhabdus/genética
8.
Emerg Microbes Infect ; 8(1): 707-716, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31119985

RESUMO

Staphylococcus aureus (S. aureus) is one of the most serious human pathogens. α-Hemolysin (Hla) secreted by S. aureus is a key toxin for various infections. We herein report that Honokiol, a natural plant polyphenol, inhibits the secretion and hemolytic activity of staphylococcal Hla with concomitant growth inhibition of S. aureus and protection of S. aureus-mediated cell injury within subinhibitory concentrations. In parallel, Honokiol attenuates the staphylococcal Hla-induced inflammatory response by inhibiting NLRP3 inflammasome activation in vitro and in vivo. Consequently, the biologically active forms of the inflammatory cytokines IL-1ß and IL-18 are reduced significantly in response to Honokiol in mice infected with S. aureus. Experimentally, we confirm that Honokiol binds to monomeric Hla with a modest affinity without impairing its oligomerization. Based on molecular docking analyses in silico, we make a theoretical discovery that Honokiol is located outside of the triangular region of monomeric Hla. The binding model restricts the function of the residues related to membrane channel formation, which leads to the functional disruption of the assembled membrane channel. This research creates a new paradigm for developing therapeutic agents against staphylococcal Hla-mediated infections.


Assuntos
Toxinas Bacterianas/metabolismo , Compostos de Bifenilo/administração & dosagem , Proteínas Hemolisinas/metabolismo , Inflamassomos/antagonistas & inibidores , Lignanas/administração & dosagem , Receptores de Superfície Celular/antagonistas & inibidores , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Células A549 , Animais , Antibacterianos/administração & dosagem , Antibacterianos/farmacologia , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Toxinas Bacterianas/toxicidade , Compostos de Bifenilo/metabolismo , Compostos de Bifenilo/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Proteínas Hemolisinas/toxicidade , Histocitoquímica , Humanos , Lignanas/metabolismo , Lignanas/farmacologia , Fígado/patologia , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Ligação Proteica , Infecções Estafilocócicas/patologia , Staphylococcus aureus/metabolismo , Resultado do Tratamento
9.
Int J Mol Sci ; 20(9)2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31052154

RESUMO

The SpyCatcher-SpyTag system was developed seven years ago as a method for protein ligation. It is based on a modified domain from a Streptococcus pyogenes surface protein (SpyCatcher), which recognizes a cognate 13-amino-acid peptide (SpyTag). Upon recognition, the two form a covalent isopeptide bond between the side chains of a lysine in SpyCatcher and an aspartate in SpyTag. This technology has been used, among other applications, to create covalently stabilized multi-protein complexes, for modular vaccine production, and to label proteins (e.g., for microscopy). The SpyTag system is versatile as the tag is a short, unfolded peptide that can be genetically fused to exposed positions in target proteins; similarly, SpyCatcher can be fused to reporter proteins such as GFP, and to epitope or purification tags. Additionally, an orthogonal system called SnoopTag-SnoopCatcher has been developed from an S. pneumoniae pilin that can be combined with SpyCatcher-SpyTag to produce protein fusions with multiple components. Furthermore, tripartite applications have been produced from both systems allowing the fusion of two peptides by a separate, catalytically active protein unit, SpyLigase or SnoopLigase. Here, we review the current state of the SpyCatcher-SpyTag and related technologies, with a particular emphasis on their use in vaccine development and in determining outer membrane protein localization and topology of surface proteins in bacteria.


Assuntos
Toxinas Bacterianas/química , Engenharia de Proteínas/métodos , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Nanopartículas/química , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptococcus pyogenes/química , Streptococcus pyogenes/metabolismo
10.
Bull Exp Biol Med ; 166(6): 759-765, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31028588

RESUMO

Campylobacter genus bacteria causing campylobacteriasis are difficult to culture. This fact necessitates creation of special approaches to studies of the behavior of these pathogens during the manufacture and storage of foodstuffs. The regularities of Campylobacter jejuni transition into an uncultivable state are studied under conditions simulating the process of immersion cooling of fresh poultry products. The proportion of viable colony-forming (CFU) cells to the total count of planktonic and uncultivable cells in the population was calculated by the level of genomic DNA in the samples evaluated by quantitative real-time PCR with intercalating dyes. PCR was carried out with primers detecting the cytolethal toxin subunit B gene cdtB and invasion gene ciaB in C. jejuni strains. The count of detected cells was 5-10-fold higher than the count of CFU; the cultural method failed to detect the agent in 40% analyzed samples of superficially infected products, while the level of uncultivable cells detected by PCR was significantly higher. The relationship between culturing conditions and formation of C. jejuni biofilms was studied. The most intensive formation of film exomatrix was observed under unfavorable conditions for this microorganism at 25oC. In microaerophilic gaseous medium, weak formation of films and intensive growth of C. jejuni populations were observed. Culturing at higher temperatures (37-42oC) was in fact inessential for the film formation process.


Assuntos
Antígenos de Bactérias/genética , Toxinas Bacterianas/genética , Biofilmes/crescimento & desenvolvimento , Campylobacter jejuni/crescimento & desenvolvimento , Produtos Avícolas/microbiologia , Animais , Antígenos de Bactérias/isolamento & purificação , Antígenos de Bactérias/metabolismo , Carga Bacteriana , Toxinas Bacterianas/isolamento & purificação , Toxinas Bacterianas/metabolismo , Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , Galinhas , Contagem de Colônia Microbiana , Contaminação de Alimentos/análise , Expressão Gênica , Humanos , Reação em Cadeia da Polimerase , Temperatura Ambiente
11.
PLoS Pathog ; 15(4): e1007704, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30951565

RESUMO

Typhoid toxin is a virulence factor for Salmonella Typhi and Paratyphi, the cause of typhoid fever in humans. This toxin has a unique architecture in that its pentameric B subunit, made of PltB, is linked to two enzymatic A subunits, the ADP ribosyl transferase PltA and the deoxyribonuclease CdtB. Typhoid toxin is uniquely adapted to humans, recognizing surface glycoprotein sialoglycans terminated in acetyl neuraminic acid, which are preferentially expressed by human cells. The transport pathway to its cellular targets followed by typhoid toxin after receptor binding is currently unknown. Through a genome-wide CRISPR/Cas9-mediated screen we have characterized the mechanisms by which typhoid toxin is transported within human cells. We found that typhoid toxin hijacks specific elements of the retrograde transport and endoplasmic reticulum-associated degradation machineries to reach its subcellular destination within target cells. Our study reveals unique and common features in the transport mechanisms of bacterial toxins that could serve as the bases for the development of novel anti-toxin therapeutic strategies.


Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Degradação Associada com o Retículo Endoplasmático , Salmonella typhi/patogenicidade , Febre Tifoide/microbiologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Transporte Biológico , Sistemas CRISPR-Cas , Células HeLa , Humanos , Espaço Intracelular/metabolismo , Ligação Proteica , Salmonella typhi/genética , Febre Tifoide/genética , Febre Tifoide/metabolismo
12.
MBio ; 10(2)2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015325

RESUMO

The cholesterol-dependent cytolysin (CDC) genes are present in bacterial species that span terrestrial, vertebrate, and invertebrate niches, which suggests that they have evolved to function under widely different environmental conditions. Using a combination of biophysical and crystallographic approaches, we reveal that the relative stability of an intramolecular interface in the archetype CDC perfringolysin O (PFO) plays a central role in regulating its pore-forming properties. The disruption of this interface allows the formation of the membrane spanning ß-barrel pore in all CDCs. We show here that the relative strength of the stabilizing forces at this interface directly impacts the energy barrier posed by the transition state for pore formation, as reflected in the Arrhenius activation energy (Ea) for pore formation. This change directly impacts the kinetics and temperature dependence of pore formation. We further show that the interface structure in a CDC from a terrestrial species enables it to function efficiently across a wide range of temperatures by minimizing changes in the strength of the transition state barrier to pore formation. These studies establish a paradigm that CDCs, and possibly other ß-barrel pore-forming proteins/toxins, can evolve significantly different pore-forming properties by altering the stability of this transitional interface, which impacts the kinetic parameters and temperature dependence of pore formation.IMPORTANCE The cholesterol-dependent cytolysins (CDCs) are the archetype for the superfamily of oligomeric pore-forming proteins that includes the membrane attack complex/perforin (MACPF) family of immune defense proteins and the stonefish venom toxins (SNTX). The CDC/MACPF/SNTX family exhibits a common protein fold, which forms a membrane-spanning ß-barrel pore. We show that changing the relative stability of an extensive intramolecular interface within this fold, which is necessarily disrupted to form the large ß-barrel pore, dramatically alters the kinetic and temperature-dependent properties of CDC pore formation. These studies show that the CDCs and other members of the CDC/MACPF/SNTX superfamily have the capacity to significantly alter their pore-forming properties to function under widely different environmental conditions encountered by these species.


Assuntos
Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Toxinas Bacterianas/genética , Fenômenos Químicos , Cristalografia por Raios X , Análise Mutacional de DNA , Proteínas Hemolisinas/genética , Cinética , Simulação de Dinâmica Molecular , Proteínas Citotóxicas Formadoras de Poros/genética , Temperatura Ambiente
13.
Microbiol Spectr ; 7(2)2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30873936

RESUMO

Staphylococcus aureus is a formidable pathogen capable of causing infections in different sites of the body in a variety of vertebrate animals, including humans and livestock. A major contribution to the success of S. aureus as a pathogen is the plethora of virulence factors that manipulate the host's innate and adaptive immune responses. Many of these immune modulating virulence factors are secreted toxins, cofactors for activating host zymogens, and exoenzymes. Secreted toxins such as pore-forming toxins and superantigens are highly inflammatory and can cause leukocyte cell death by cytolysis and clonal deletion, respectively. Coagulases and staphylokinases are cofactors that hijack the host's coagulation system. Exoenzymes, including nucleases and proteases, cleave and inactivate various immune defense and surveillance molecules, such as complement factors, antimicrobial peptides, and surface receptors that are important for leukocyte chemotaxis. Additionally, some of these secreted toxins and exoenzymes can cause disruption of endothelial and epithelial barriers through cell lysis and cleavage of junction proteins. A unique feature when examining the repertoire of S. aureus secreted virulence factors is the apparent functional redundancy exhibited by the majority of the toxins and exoenzymes. However, closer examination of each virulence factor revealed that each has unique properties that have important functional consequences. This chapter provides a brief overview of our current understanding of the major secreted virulence factors critical for S. aureus pathogenesis.


Assuntos
Toxinas Bacterianas/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo , Animais , Toxinas Bacterianas/classificação , Toxinas Bacterianas/imunologia , Humanos , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/enzimologia , Staphylococcus aureus/imunologia
14.
Science ; 364(6435): 82-85, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30872531

RESUMO

Intracellular pathogens and danger signals trigger the formation of inflammasomes, which activate inflammatory caspases and induce pyroptosis. The anthrax lethal factor metalloprotease and small-molecule DPP8/9 inhibitors both activate the NLRP1B inflammasome, but the molecular mechanism of NLRP1B activation is unknown. In this study, we used genome-wide CRISPR-Cas9 knockout screens to identify genes required for NLRP1B-mediated pyroptosis. We discovered that lethal factor induces cell death via the N-end rule proteasomal degradation pathway. Lethal factor directly cleaves NLRP1B, inducing the N-end rule-mediated degradation of the NLRP1B N terminus and freeing the NLRP1B C terminus to activate caspase-1. DPP8/9 inhibitors also induce proteasomal degradation of the NLRP1B N terminus but not via the N-end rule pathway. Thus, N-terminal degradation is the common activation mechanism of this innate immune sensor.


Assuntos
Antígenos de Bactérias/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Toxinas Bacterianas/metabolismo , Inflamassomos/metabolismo , Proteólise , Piroptose/fisiologia , Animais , Proteínas Reguladoras de Apoptose/genética , Sistemas CRISPR-Cas , Caspase 1/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/antagonistas & inibidores , Técnicas de Inativação de Genes , Células HEK293 , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Piroptose/genética , Células RAW 264.7 , Inibidores de Serino Proteinase/farmacologia , Células THP-1 , Ubiquitina-Proteína Ligases/genética
15.
Appl Microbiol Biotechnol ; 103(8): 3341-3353, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30887174

RESUMO

Antigen-binding fragments (Fabs) are an important part of monoclonal antibody (mAb) therapeutics and can be cost-effectively produced using an Escherichia coli (E. coli) expression system. However, Fabs tend to form undesirable aggregates when expressed in the cytoplasm of E. coli, substantially reducing the yield of correctly folded proteins. To solve this problem, in this study, we used five Fab fragments targeting IGF1R, Her2, VEGF, RANKL, and PD-1 to develop a novel system employing the alkaline phosphatase (phoA) promoter and the heat-stable enterotoxin II (STII) leader sequence to facilitate the efficient expression and extracellular secretion of Fabs. Following phosphate starvation, all five Fab fragments were expressed in BL21(DE3), were largely secreted into the culture medium, and then, were further purified by affinity chromatography specific to the constant region of the light chain. The purified Fab products were evaluated and were found to have high purity, antigen-binding affinity, and in vitro bioactivity. The mechanism experiments revealed that (1) BL21(DE3) had significantly higher productivity than the K-12 strains investigated; (2) the secretion ability of the PhoA promoter was superior to that of the T7 promoter; and (3) signal peptide, STII, showed higher extracellular secretion efficiency than pelB. Our findings strongly suggested that the phoA-STII-facilitated extracellular production platform is highly promising for application in the manufacturing of Fab fragments for both academic and industrial purposes.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos Fab das Imunoglobulinas/metabolismo , Fosfatase Alcalina/genética , Afinidade de Anticorpos , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Meios de Cultura/química , Enterotoxinas/genética , Enterotoxinas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Regiões Promotoras Genéticas , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
16.
Nat Commun ; 10(1): 972, 2019 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-30814507

RESUMO

Bacterial toxin-antitoxin (TA) modules are tightly regulated to maintain growth in favorable conditions or growth arrest during stress. A typical regulatory strategy involves the antitoxin binding and repressing its own promoter while the toxin often acts as a co-repressor. Here we show that Pseudomonas putida graTA-encoded antitoxin GraA and toxin GraT differ from other TA proteins in the sense that not the antitoxin but the toxin possesses a flexible region. GraA auto-represses the graTA promoter: two GraA dimers bind cooperatively at opposite sides of the operator sequence. Contrary to other TA modules, GraT is a de-repressor of the graTA promoter as its N-terminal disordered segment prevents the binding of the GraT2A2 complex to the operator. Removal of this region restores operator binding and abrogates Gr aT toxicity. GraTA represents a TA module where a flexible region in the toxin rather than in the antitoxin controls operon expression and toxin activity.


Assuntos
Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Antitoxinas/genética , Antitoxinas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/toxicidade , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Genes Bacterianos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Óperon , Regiões Promotoras Genéticas , Ligação Proteica , Dobramento de Proteína , Estrutura Quaternária de Proteína , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Eletricidade Estática , Sistemas Toxina-Antitoxina/genética
17.
MBio ; 10(2)2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30890614

RESUMO

Mucosal and skin tissues form barriers to infection by most bacterial pathogens. Staphylococcus aureus causes diseases across these barriers in part dependent on the proinflammatory properties of superantigens. We showed, through use of a CRISPR-Cas9 CD40 knockout, that the superantigens toxic shock syndrome toxin 1 (TSST-1) and staphylococcal enterotoxins (SEs) B and C stimulated chemokine production from human vaginal epithelial cells (HVECs) through human CD40. This response was enhanced by addition of antibodies against CD40 through an unknown mechanism. TSST-1 was better able to stimulate chemokine (IL-8 and MIP-3α) production by HVECs than SEB and SEC, suggesting this is the reason for TSST-1's exclusive association with menstrual TSS. A mutant of TSST-1, K121A, caused TSS in a rabbit model when administered vaginally but not intravenously, emphasizing the importance of the local vaginal environment. Collectively, our data suggested that superantigens facilitate infections by disruption of mucosal barriers through their binding to CD40, with subsequent expression of chemokines. The chemokines facilitate TSS and possibly other epithelial conditions after attraction of the adaptive immune system to the local environment.IMPORTANCE Menstrual toxic shock syndrome (TSS) is a serious infectious disease associated with vaginal colonization by Staphylococcus aureus producing the exotoxin TSS toxin 1 (TSST-1). We show that menstrual TSS occurs after TSST-1 interaction with an immune costimulatory molecule called CD40 on the surface of vaginal epithelial cells. Other related toxins, where the entire family is called the superantigen family, bind to CD40, but not with a high-enough apparent affinity to cause TSS; thus, TSST-1 is the only exotoxin superantigen associated. Once the epithelial cells become activated by TSST-1, they produce soluble molecules referred to as chemokines, which in turn facilitate TSST-1 activation of T lymphocytes and macrophages to cause the symptoms of TSS. Identification of small-molecule inhibitors of the interaction of TSST-1 with CD40 may be useful so that they may serve as additives to medical devices, such as tampons and menstrual cups, to reduce the incidence of menstrual TSS.


Assuntos
Antígenos CD40/metabolismo , Quimiocinas/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Staphylococcus aureus/fisiologia , Superantígenos/metabolismo , Toxinas Bacterianas/metabolismo , Antígenos CD40/genética , Células Cultivadas , Enterotoxinas/metabolismo , Técnicas de Inativação de Genes , Humanos
18.
Science ; 364(6435)2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30872533

RESUMO

Inflammasomes are multiprotein platforms that initiate innate immunity by recruitment and activation of caspase-1. The NLRP1B inflammasome is activated upon direct cleavage by the anthrax lethal toxin protease. However, the mechanism by which cleavage results in NLRP1B activation is unknown. In this study, we find that cleavage results in proteasome-mediated degradation of the amino-terminal domains of NLRP1B, liberating a carboxyl-terminal fragment that is a potent caspase-1 activator. Proteasome-mediated degradation of NLRP1B is both necessary and sufficient for NLRP1B activation. Consistent with our functional degradation model, we identify IpaH7.8, a Shigella flexneri ubiquitin ligase secreted effector, as an enzyme that induces NLRP1B degradation and activation. Our results provide a unified mechanism for NLRP1B activation by diverse pathogen-encoded enzymatic activities.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antígenos de Bactérias/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Inflamassomos/imunologia , Peptídeo Hidrolases/metabolismo , Proteólise , Shigella flexneri/patogenicidade , Ubiquitina-Proteína Ligases/metabolismo , Animais , Bacillus anthracis/enzimologia , Toxinas Bacterianas/metabolismo , Proteínas Adaptadoras de Sinalização CARD/química , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Caspase 1/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/química , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Ativação Enzimática , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Domínios Proteicos , Subunidades Proteicas , Células RAW 264.7 , Shigella flexneri/enzimologia
19.
World J Microbiol Biotechnol ; 35(3): 52, 2019 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-30868269

RESUMO

Several Bacillus strains are used as biocontrol agents, as they frequently have strong antagonistic effects against microbial plant pathogens. Bacillus strain SZMC 6179J, isolated from tomato rhizosphere, was previously shown to have excellent in vitro antagonistic properties against the most important fungal pathogens of tomato (Alternaria solani, Botrytis cinerea, Phytophthora infestans and Sclerotinia sclerotiorum) as well as several Fusarium species. Taxonomic investigations revealed that it is a member of the B. subtilis subsp. subtilis group and very closely related with the reference type strain B. subtilis subsp. subtilis 168. The sequenced genome of strain SZMC 6179J contains the genes responsible for the synthesis of the extracellular antibiotics surfactin, fengycin and bacilysin. Compared to strain 168, a prophage-like region is missing from the genome of SZMC 6179J, while there are 106 single nucleotide polymorphisms and 23 deletion-insertion polymorphisms. The high biocontrol potential of strain SZMC 6179J may results from a single base deletion in the sfp gene encoding the transcription factor of the surfactin and fengycin operons. Hypermutated regions reflecting short-time evolutionary processes could be detected in SZMC 6179J. The deletion-insertion polymorphism in the sfp gene and the detected hypermutations can be suggested as genetic determinants of biocontrol features in B. subtilis.


Assuntos
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Agentes de Controle Biológico/metabolismo , Mutação , Sequência de Aminoácidos , Antibacterianos/metabolismo , Antifúngicos/metabolismo , Bacillus subtilis/classificação , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Dipeptídeos/genética , Dipeptídeos/metabolismo , Fungos/efeitos dos fármacos , Fungos/patogenicidade , Genes Bacterianos/genética , Lipopeptídeos/genética , Lipopeptídeos/metabolismo , Lycopersicon esculentum/microbiologia , Família Multigênica , Tipagem de Sequências Multilocus , Peptídeos Cíclicos/genética , Filogenia , Doenças das Plantas/microbiologia , Polimorfismo de Nucleotídeo Único , Metabolismo Secundário/genética , Fatores de Transcrição/genética , Sequenciamento Completo do Genoma
20.
J Ind Microbiol Biotechnol ; 46(6): 869-882, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30805740

RESUMO

Bacterial toxin-antitoxin (TA) systems play important roles in diverse cellular regulatory processes. Here, we characterize three putative type II TA candidates from Acetobacter pasteurianus and investigate the profile of type II TA systems in the genus Acetobacter. Based on the gene structure and activity detection, two-pairs loci were identified as the canonical hicAB and higAB TA systems, respectively, and DB34_01190-DB34_01195 as a putative new one without a canonical TA architecture. Physiologically, the expression of the three pairs conferred E. coli with additional plasmid maintenance and survival when under acetic acid stress. Chromosomal TA systems can be horizontally transferred within an ecological vinegar microbiota by co-option, and there was a tendency for toxin module loss. The antitoxin retention in the genome is suggested to have a broad role in bacterial physiology. Furthermore, A. pasteurianus strains, universally domesticated and used for industrial vinegar fermentation, showed a higher number of type II TA loci compared to the host-associated ones. The amount of TA loci per genome showed little positive relationship to insertion sequences, although its prevalence was species-associated, to the extent of even being strain-associated. The TA system is a candidate of studying the resistant mechanistic network, the TAs-dependent translatome affords a real-time profile to explore stress adaptation of A. pasteurianus, promoting industrial development.


Assuntos
Acetobacter/metabolismo , Toxinas Bacterianas/metabolismo , Fermentação , Sistemas Toxina-Antitoxina/fisiologia , Ácido Acético/metabolismo , Adaptação Fisiológica , Fermentação/genética , Fermentação/fisiologia , Genoma Bacteriano/genética , Genômica
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